Method and system implementing spatially modulated excitation or emission for particle characterization with enhanced sensitivity

ABSTRACT

A method and system for using spatially modulated excitation/emission and relative movement between a particle (cell, molecule, aerosol, . . . ) and an excitation/emission pattern are provided. In at least one form, an interference pattern of the excitation light with submicron periodicity perpendicular to the particle flow is used. As the particle moves along the pattern, emission is modulated according to the speed of the particle and the periodicity of the stripe pattern. A single detector, which records the emission over a couple of stripes, can be used. The signal is recorded with a fast detector read-out in order to capture the “blinking” of the particles while they are moving through the excitation pattern. This concept enables light detection with high signal-to-noise ratio and high spatial resolution without the need of expensive and bulky optics.

CROSS REFERENCE TO RELATED PATENTS AND APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/698,409 filed Jan.26, 2007, now U.S. Pat. No. 8,821,799, which is incorporated herein byreference in its entirety. This application is related to U.S.application Ser. No. 11/698,338 filed on Jan. 26, 2007.

BACKGROUND

Methods for particle characterization (which generally relates todetection as well as other useful characterizations such aslocation/position determination, particle counting and cell sorting)often suffer from a low signal-to-noise ratio (SNR), since the signalobtained from the particle (in general: a small object) is typicallyweak in comparison to the background. This is particularly true inconnection with optical methods of particle characterization. The lowsignal-to-noise ratio is also particularly noteworthy in cases ofdetection of individual particles such as a cell, an aerosol, amolecule, a subvolume of liquid which differs from the surroundingliquid or emulsion, or a piece of DNA with dyes or tags at selectionpositions.

With respect to the DNA case, conventional DNA sequencing isaccomplished by splitting a DNA strand into small pieces, separating thepieces with electrophoresis and then elaborately reconstructing the DNAsequence. An alternative process has recently been developed. In thisalternative process, certain base sequences are tagged with fluorescentdyes. After stretching (or “linearizing”) the molecule, the DNA strandis moved through a microfluidic channel at a constant speed. A specialfluorescence reader with a high spatial resolution (approx. 1 μm) isused to record the positions of the fluorescent dyes or tags. As aresult, an “optical bar code” of the DNA containing the position of thetags is recorded. Therefore, the DNA sequence may be identified.

Typical distance between the tags along the DNA is several μm.Consequently, the required spatial resolution is one μm or better.Typically, this concept is demonstrated by using a con-focal microscope,which allows for exciting and also detecting the fluorescence within avery small volume (−1 μm3).

FIG. 1 schematically illustrates a conventional approach for spatiallyresolved fluorescence excitation. As shown, a system 10 includes adetector 12, a channel 14 and an excitation light 16. A small volumewithin the channel 14 is excited. Light is collected from the excitedvolume. DNA strings 20 with tagged portions 22 run through an excitationarea 24 of the channel 14. The positions of the tags are calculatedusing a time dependent detector signal.

This approach has been successfully implemented. However, it requiressophisticated and bulky optics to ensure suitably sized excitation anddetection volumes. Moreover, the resultant signal-to-noise ratios arelower than desired.

INCORPORATION BY REFERENCE

U.S. Pat. No. 9,164,037 is hereby incorporated herein by reference inits entirety.

BRIEF DESCRIPTION

In one aspect of the presently described embodiments, the methodcomprises generating a spatially modulated excitation region, creatingrelative movement between a particle and the excitation region, theparticle being excited upon exposure to the excitation region to obtaina time modulated signal, and, recording the modulated signal.

In another aspect of the presently described embodiments, the excitationregion includes an excitation pattern.

In another aspect of the presently described embodiments, the timemodulated signal is caused by light emission from the particle.

In another aspect of the presently described embodiments, the excitationregion comprises interference stripes.

In another aspect of the presently described embodiments, the excitationregion is generated by at least one of a shadow mask and a lens array.

In another aspect of the presently described embodiments, the excitationpattern is generated by chemo-luminescence.

In another aspect of the presently described embodiments, the methodfurther comprises at least one of determining a location of the particlebased on the signal, counting particles based on the signal, and sortingparticles based on the signal.

In another aspect of the presently described embodiments, the detectingcomprises detecting with a pixilated detector.

In another aspect of the presently described embodiments, the particleis a portion of a DNA molecule or a molecule attached to the DNAmolecule and the signal is used to determine DNA sequencing.

In another aspect of the presently described embodiments, the detectingcomprises using a spectrometer to receive the fluorescent spectrum ofthe fluorescing analyte.

In another aspect of the presently described embodiments, the generatingof the excitation region comprises generating a spatially modulatedpattern based on at least one of geometry, electric or magnetic field,fluorescence quenching, analyte concentration, density, and acousticstanding wave.

In another aspect of the presently described embodiments, the generatingof the excitation pattern comprises generating a spatially modulatedregion based on environment.

In another aspect of the presently described embodiments, thegenerating, creating and recording is conducted in two-dimensions tolocate the particle.

In another aspect of the presently described embodiments, a method forcharacterizing particles comprises moving a particle within a channel,providing an environment along the channel which causes the particle tocreate a time modulated signal, and, detecting and evaluating the timemodulated signal.

In another aspect of the presently described embodiments, theenvironment comprises an optical element and the particle emits lightdetected by the optical element.

In another aspect of the presently described embodiments, the opticalelement is operative to modulate the signal obtained from the particleas a function of a position of a particle.

In another aspect of the presently described embodiments, the methodfurther comprises moving the optical element.

In another aspect of the presently described embodiments, the opticalelement is one of a shadow mask and a micro-lens array.

In another aspect of the presently described embodiments, the methodfurther comprises at least one of determining a location of the particlebased on the signal, counting particles based on the signal, and sortingparticles based on the signal.

In another aspect of the presently described embodiments, the detectingcomprises detecting with a pixilated detector.

In another aspect of the presently described embodiments, the particleis a portion of a DNA molecule or a molecule attached to the DNAmolecule and the signal is used to determine DNA sequencing.

In another aspect of the presently described embodiments, the detectingcomprises using a spectrometer to receive a fluorescent spectrum of theparticle.

In another aspect of the presently described embodiments, the opticalelement is operative to pattern the light based on at least one ofgeometry, electric or magnetic field, fluorescence quenching, particleconcentration, density, and acoustic standing wave.

In another aspect of the presently described embodiments, a system forcharacterizing particles comprises means for generating a spatiallymodulated excitation region, means for providing relative movementbetween a particle and the region, the particle being excited uponexposure to the excitation area to obtain a time modulated signal, meansfor recording the modulated signal.

In another aspect of the presently described embodiments, the systemfurther comprises at least one of a means for determining a location ofthe particle based on the signal a means for counting particles based onthe signal, and means for sorting particles based on the signal.

In another aspect of the presently described embodiments, a system forcharacterizing particles comprises a channel, a means for moving aparticle within the channel, an environment along the channel operativeto cause the particle to create time modulated signal, and, a detectionsystem to record and evaluate the time modulated signal.

In another aspect of the presently described embodiments, theenvironment comprises an optical element.

In another aspect of the presently described embodiments, the systemfurther comprises an anti-resonant waveguide operative to cause theparticle to emit light.

In another aspect of the presently described embodiments, the systemfurther comprises at least one of a means for determining a location ofthe fluorescent analyte based on the signal, a means for countingparticles based on the signal, and a means for sorting particles basedon the signal.

In another aspect of the presently described embodiments, theenvironment allows for a two-dimensional evaluation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representative view of a prior art system;

FIG. 2 is a representative view of a presently described embodiment;

FIG. 3 is a representative view of a presently described embodiment;

FIG. 4 is a representative view of a presently described embodiment;

FIG. 5 is a representative view of a presently described embodiment;

FIG. 6 is a representative view of a presently described embodiment;

FIG. 7 is a representative view of a presently described embodiment;

FIGS. 8(a)-(d) are representative views of presently describedembodiments;

FIG. 9 is a flow chart according to the presently described embodiments;

FIG. 10 is a representative view of a presently described embodiment;

FIG. 11 is a representative view of a presently described embodiment;

FIG. 12 is an illustration of various forms of signals that may be usedin connection with the presently described embodiments;

FIG. 13 is a representative view of a presently described embodiment;

FIG. 14 is a representative view of a presently described embodiment;and,

FIG. 15 is a representative view of a presently described embodiment.

DETAILED DESCRIPTION

This patent application describes a method and a system to improve thesignal-to-noise ratio during particle characterization by implementing aphase sensitive technique which allows for clearly distinguishingbetween the signals from the particle and the background. The method isbased on the deliberate introduction of controlled relative movementbetween the particle and the environment. The combination of, forexample, the moving particle and a conditioned environment results in atime modulated signal. A monitored response comprises a noisy backgroundsignal and the modulated signal, with a well defined time dependenceattributable to the particle. Both hardware and software solutions canbe applied to extract the signal attributed to the particle.

It should be understood that the signal attributed to the particle canthen be used in characterizing the particle for a variety of differentapplications, including detection, location/position determination, andcell sorting. Various techniques may be employed to accomplish thesecharacterizations. It should be noted, however, that one technique forcharacterization, i.e., determination of particle positions anddistances, is described in U.S. Pat. No. 9,164,037, and may beadvantageously implemented along with the presently describedembodiments.

It will be appreciated that the contemplated signal can have any shapeas a function of time. It is not necessarily periodic. Even a signalmodulated randomly is useful, as long as the structure of the signal isknown. In this regard, the structure may be known but the signal may notfollow an analytic regularity. So, the time periods defining “on” and“off” states for the particle will have a random length. Even where thetime dependence of the signal is built into the system, the timedependence of the system can be varied, as long as it is predictable orknown.

Note that different encodings of the signal (e.g., chirped or strictlyperiodic) carry specific advantages for a particular application.Chirped signals improve spatial resolution and particle separation.Strictly periodic signals allow for determining particle speed and aremore adaptive to particles with different speeds.

To explain, with reference to FIG. 12, various types of signals areillustrated. For example, signal 12-1 is a periodic signal. Signal 12-2is a chirped signal, e.g., a signal that has a linearly increasingfrequency. Also shown is a random signal 12-3. It should be appreciatedthat these signals (as well as others) may be used in connection withthe presently described embodiments to achieve the objectives of thepresently described embodiments.

The presently described embodiments are described for a variety of casesincluding: (1) a collection of individual moving particles (2) alinearized DNA strand in which the objects of interest are distributedand fixed along the length of the strand i.e., commonly termed DNAsequencing, and (3) a collection of particles potentially fixed on asurface (providing a need, in some applications, for a two-dimensionalanalysis).

In this regard, the particles being detected may include cells,aerosols, DNA material pieces of DNA with dyes at selected positions,subvolumes in a liquid which differs from the surrounding liquid oremulsion, droplets, other small volumes of fluid, bubbles, singlemolecules, agglomerated molecules, molecule clusters, biological cells,viruses, bacteria, proteins, microparticles, nanoparticles, andemulsions. A droplet or small volume of fluid may, for example, includeatoms, molecules or other particles that affect refractive index,absorption, or other optical characteristics. An object “travels” or iscaused “to travel” if the object moves through a succession ofpositions. For example, the object could be conveyed in a fluid, such asa liquid, gas, or aerosol, in which case the object may be referred toas being “carried by the fluid.” Still further, it should be appreciatedthat a channel may be defined in a variety of manners to include notonly ones defined by walls, but also ones defined by the flow ofparticles in, for example, an aerosol stream or the like.

It should be further understood that light emission from these particlesmay result from a variety of sources including fluorescence excitation,elastic and inelastic light scattering, chemo-luminescence or othertypes of light scattering and reflection techniques. Still further, thelight used in these implementations may include a variety of differenttypes of lighting including, ultraviolet, VIS, infrared, near infrared,etc. As will be described in greater detail below, the environments inwhich this particle characterization process is implemented includeenvironments wherein there is a spatially modulated excitation of theparticles or a modulation of an emitted light from particles over adetection region. In this regard, the particles may emit a homogeneousexcitation that is filtered using, for example, a shadow mask or otheroptics, which leads to a spatial modulation of the emitted light.

It should also be understood that the presently described embodimentsmay be applied to optical as well as non-optical environments such asthose involving capacitance, induction, resistivity, etc. FIGS. 2-14generally relate to optical cases while FIG. 15 relates to an examplenon-optical case.

With reference now to FIG. 2, a system 30 is illustrated. In the system30, a channel 34 is provided with optical elements 32 for modulatingemitted light within a detection region. The optical elements 32 maytake a variety of forms such as lenses, masks or filters. Also shown isa detector 31 for detecting the emitted light from the optical elements32 as a function of time. It should be understood that the detector 31(as well as other detectors noted herein) may take a variety of formsincluding photo-diodes, avalanche photo-diodes, photo-multiplier tubes,etc. It should also be appreciated that the detector 31 and opticalelements 32 may be combined in a single element or system.

Light may be emitted from particles such as particle 36 and particle 38that are traveling down the channel 34. It should be understood that thelight emission from the particle may result from the various phenomenondescribed above. It should also be understood that the relative movementbetween the particles 36 and 38 and the optical element system 32, oroutput modulator, create the modulation desired to be able toappropriately analyze the particles 36 and 38. The spatially modulatedoptics create a time modulated signal in the detector 31. This signal,which as noted may take a variety of predictable forms, may be analyzedusing the processing module 39 for purposes of characterizing theparticles.

As noted, the signal generated as a function of time may take a varietyof forms, e.g., periodic, chirped, random . . . etc., as a function of avariety of environmental factors. In one form, that may be applied tothis embodiment as well as the embodiments of FIGS. 3 and 4, the opticsmay take a form that coincides with the type of signal. So, the opticalelement structure may resemble that of the signal examples of FIG. 12.

It should be appreciated that the relative movement may be created byway of the particle moving, the detector/optical elements moving along,for example, the channel or by way of movement of both of theseelements. It should be further understood that movement of both of theelements may, in one form, result in movement of each element atdifferent velocities.

With reference now to FIG. 3, a system 40 is shown. The system 40includes a channel 42 that is provided with regions 44 where particles,such as particles 46 and 48, are caused to emit light. Regions 44, asshown, create a modulated excitation area that can be achieved byvarious methods. These methods include interference, use of shadowmasking, use of lens arrays and use of chemo-luminescence andfluorescent quenching. Relative movement between the particle and theexcitation area or pattern can be achieved by moving the particle, theexcitation pattern or both. As with the previous embodiment, it shouldbe appreciated that movement of both, in one form, would result inmovement of each at different speeds. The spatially modulated opticscreate a time modulated signal in the detector 41. This signal, which asnoted may take a variety of predictable forms, may be analyzed using theprocessing module 49 for purposes of characterizing the particles.

With reference now to FIG. 4, a system 50 is illustrated. System 50includes a channel 52 and an anti-resonant wave guide 54. Theanti-resonant wave guide provides homogeneous illumination of thechannel as the particle travels therethrough. Modulation can be createdby mirrors 56 which can be suitably placed inside or outside thechannel. Again, the spatially modulated optics create a time modulatedsignal in the detector 51. This signal, which as noted may take avariety of predictable forms, may be analyzed using the processingmodule 59 for purposes of characterizing the particles.

It should be apparent from the embodiments described in connection withFIGS. 2-4, the presently described embodiments provide for acharacterization of particles traveling through other types of medium(e.g., liquid) that is improved by way of modulation. In some cases, thelight emitted from particles is modulated and, in other cases, themodulation occurs by way of modulated excitation of the particles. Inboth cases, it is the relative movement of the system elements thatprovides the modulated effect.

With reference to FIG. 11, another embodiment is illustrated. Of course,various configurations involving the imaging of a particle to a detectorplane are contemplated. However, one such example as shown includes asystem 300 positioned near a channel 302 in which a particle 304 istraveling. The particle 304 is detected by a detector 306 having anoptical element 308 positioned thereon operative to produce a spatialpattern. The detection of the particle is facilitated by optics 310,which may take a variety of forms including a lens or lens system. Theparticle lies in an object plane 320 while an image plane 322 isassociated with the detector 306 and optical element 308.

In this arrangement, the particle size, pattern size and spatialresolution are essentially de-coupled. In this regard, the optics 310serve to magnify (or de-magnify) the particle 304 and conduct thedetecting at a location remote from the particle 304. As shown, lightoriginating from the particle 304 is modulated in the image plane 322.The detector 306 is then able to detect the light from the particle 304in the channel 302 without being positioned on the channel 302. Usingthis configuration, the optical element 308 should be in or near theimage plane 322 of the optics 310. In this way, the “optical distance”between the particle 304 and the optical element 308 is minimized. Itshould be appreciated that the detector itself can contain optics aswell as introduce additional magnification or de-magnification. Inaddition, the size of the detector is a factor in the sampling rate ofthe system. In some cases it might therefore be preferable to de-magnifythe channel on a small and faster detector to gain increased samplingrate.

Further, the optical element may be positioned on the channel itself. Ifso, the distance between the detector and the optical element would bedetermined by the channel dimensions.

A more specific implementation of the presently described embodimentsrelates to DNA sequencing. With reference to FIG. 5, a system 100according to the presently described embodiments is shown. The systemincludes a detector 102, a fluid channel 104 and excitation light 106having a spatially modulated excitation pattern 108 therewithin. Thepattern 108 aligns with the channel 104 to create a detection region orarea 105. The system 100 allows for conveyance of, for example, a DNAstring 110 having a backbone portion 112 and fluorescent tags, orparticles, 114 embedded therein. In at least one form, the interferencepattern 108 is generated by an energy source such as a laser source andhas a submicron periodicity perpendicular to particle flow. It should beappreciated that other angles might also be used. As a fluorescentparticle 114 moves down the channel 104 through the pattern 108, thefluorescent emission is modulated according to the speed of thefluorescent particle or tag 114 and the periodicity of the stripepattern 108. The signal detector 102, which records the emission over atleast one stripe can be used. The detected signal is recorded with afast detector read-out in order to capture the “blinking” of thefluorescent particle(s) or tags 114 while they are moving through thedetection area 105 and, consequently, the excitation pattern 108. Asshown, a processing module 109 may be implemented to analyze the signalfor purposes of particle characterization.

The presently described embodiments enable fluorescence detection withhigh signal-to-noise ratio and high spatial resolution, as discussed inthe following, without the need of expensive and bulky optics.

In the particular application contemplated, the DNA backbone 112 istypically labeled with one type of fluorophore and specific portions ofthe linearized molecule are labeled with a second type of fluorophore,that is, fluorescent tags 114. FIG. 6 illustrates the detection schemeaccording to the presently described embodiments in more detail. Asshown, the fluorescence backbone signal 120 first increases in astep-like function when the uniformly labeled DNA backbone 112 movesinto the detection area 105. Additionally, the backbone signal 120 issuperimposed with a sinusoidal signal 122 when one of the tags 114 movesthrough the excitation pattern 108. This results in a detected signal126.

In the exemplary embodiment illustrated in FIG. 5, only a singledetector is shown. However, multiple detectors may be implemented. Also,it should be appreciated that various configurations of detectors may beimplemented to realize the presently described embodiments.

In this regard, with reference now to FIG. 7, a system 200 wherein itmight be favorable to detect the tagged portions 114 and the DNAbackbone 112 with different detectors is shown. Backbone portions 112and tagged portions 114 are usually labeled with fluorophores emittingat different wavelength bands. Appropriate filters ensure that onlyfluorescence originating from the tags enters detector 202 whereasdetector 204 receives light only from the backbone. Simple opticalcomponents can be incorporated to improve wavelength selectivity of thefilters. For example, micro-lens arrays can collimate the fluorescentlight. The filters can be designed such that they do not only transmitthe first wavelength band of interest but also efficiently reflect thesecond wavelength band of interest onto the opposite detector in orderto minimize fluorescent light loss. The system 200 of FIG. 7 can also beused to measure the fluorescence from different fluorescence tagsmarking different portions of the DNA simultaneously.

Also, it should be appreciated that second stage detectors 206 and 208may likewise be implemented to refine, for example, the DNA analysisthat is being conducted. For example, to refine a DNA characterization,fluorescence from differently colored tags marking different portions ofa DNA strand may be measured.

To generate a suitable pattern, several well known techniques can beapplied to create, for example, an interference pattern as depicted inFIGS. 8(a)-(d). Stripes are usually generated by creating a standingwave in one dimension. For example, in FIG. 8b , a mirror 80 creates astanding wave in the channel direction. Different directions also may beused. The distance d between two adjacent interference maxima can becalculated as follows:

${d\frac{\lambda}{2\sin\;\alpha}},$

where α indicates the relative angle between the two interfering beamsand λ is the wavelength, with d varying between λ/2 and infinitydependent upon α.

In general, the excitation pattern can be directed onto the channel“from outside” through the top or bottom surface or “in plane” from theside. As the detection components are most probably attached from topand/or bottom it is favorable to use “in-plane” excitation in order toreduce the amount of excitation light that reach the detectors.

All of the interferometer techniques shown in FIG. 8(a)-(d) can berealized in the microfluidic chamber by using waveguide structures andmirrors based on total internal reflection (TIR) on substrate/airinterfaces. FIG. 8(a) illustrates a prism interferometer. FIG. 8(b)illustrates a Lloyd interferometer using the mirror 80. FIG. 8(c)illustrates a Michelson interferometer using a beam splitter 84 andmirrors 86. In order to achieve an even higher signal-to-noise ratio itwould be feasible to periodically move the interference pattern in thechannel direction. This can be achieved by mounting a mirror as shown inFIG. 8(c) onto a periodically moving stage (e.g., a speaker or piezoelement). This technique can also be applied to address stationaryparticles. This allows improvement in the sensitivity of the system byusing a double modulation technique.

Last, FIG. 8(d) illustrates a phase mask interferometer 88. A favorableapproach is based on phase masks or transmission gratings, as shown inFIG. 8(d). Light scattered into the −1st order interferes with lightscattered in +1st order. The gratings can be designed such thatscattering into 0 and 2nd order is minimized. Most techniques shown inthis section can be designed such that multiple-fluidic-channelexcitation is feasible. FIG. 10 illustrates another design forilluminating parallel channels which is based on phase masks, such asphase mask 90.

Having thus described example systems incorporating the teachings of thepresently described embodiments, it should be understood that themethods according to the presently described embodiments include, in atleast one form, the basic undertakings of creating a spatially modulatedenvironment, detecting light emitted from the excited particles in theenvironment, and generating a time modulated signal based on thedetecting. In at least one form, the generated signal is used todetermine positions of the excited particles, e.g. tags in the DNAstrand. The system, in one form, is provided with a processing module(e.g., processing modules 39, 49, 59 and 109) to allow for thegeneration of the spatially modulated signal and any subsequentprocessing such as determining the relative positions of particles. Thisprocessing module may be incorporated within the detector or implementedapart from the detector. Along these lines, it should be understood thatthe methods described herein may be implemented using a variety ofhardware configurations and software techniques. For example, thecontemplated processing module may include software routines that willallow for signal generation and location determination.

With reference now to FIG. 9, a method 500 according to at least one ofthe presently described embodiments is illustrated. Initially, anexcitation pattern is generated (at 502). As noted, the excitation maybe homogeneous with a patterned output generated by a shadow mask or thelike (e.g., a modulator). Next, relative movement between, for example,a fluorescing analyte and the excitation pattern is provided (at 504).Any emitted or scattered light as function of time is then detected (at506). A spatially modulated signal based on the detecting is thengenerated (at 508). As noted above, the spatially modulated signal canthen be used, in one form, to determine the relative positions offluorescing particles such as tags in a DNA strand.

With respect to detection of a signal relating to the fluorescentemissions as with the DNA implementation, the modulated excitation notonly ensures high spatial resolution but at the same time enables amethod to increase the signal-to-noise ratio. Most sources whichcontribute to the background signal (e.g., the backbone signal,fluorescence excited by scattered excitation, or all other DC-likesources) are eliminated by a correlation technique, which allows theread-out to be only sensitive to the modulated signal originating fromthe moving tags. Considering a tag-speed of 15 m/ms (or mm/s), an aperiodic excitation pattern with a stripe width of 1 m and a size of thetagged portion considerably less than the excitation stripe, results ina transit time of approximately 70 s per period. This results in amodulation of the fluorescence signal in the order of 10 kHz.Additionally, the excitation source can be modulated with a higherfrequency in order to separate fluorescence light from other backgroundsources (e.g., room light). The frequency has to be chosen high enoughto ensure that the light source is switched on and off several timeswhile a tag is passing one interference fringe. A modulation frequencyof 100 kHz fulfills that criterion and is easily feasible. As muchfaster detectors are available, it is even possible to applyconventional lock-in or correlation techniques to sample more accuratelyat 100 kHz, e.g., by modulating the excitation light with 1 MHz in phasewith a detector.

It should be understood that, in at least one form of the presentlydescribed embodiments, in order to determine the precise position of thetags on the DNA, the detector signal (no mater how obtained) isde-convoluted. The signal is recorded with a high sampling rate. Thetime information is thus converted to position information using thevelocity of the DNA string. In the case of a strictly periodicexcitation pattern, the velocity of the DNA string is extracted from theperiodicity of the time dependent fluorescence signal or can be measuredby other well known techniques. The analysis can be done, using avariety of signal processing algorithms including Fourier-Transformationanalysis or Least-Square fitting techniques. Some of these techniquesare described in greater detail in, for example, U.S. Pat. No. 9,164,037, which is hereby incorporated herein by reference in its entirety.

More generally, these techniques can be applied to still other kinds ofdetection for purposes of particle characterization. With reference toFIG. 13, a further embodiment of the presently described embodiments isshown. As note above, the presently described embodiments may be appliednot only in one dimension, but in two dimensions as well. In thisregard, a particle scanner 200 may be used to locate or localize aparticle 202 using a two dimensional technique. The particle 202 may bewithin a fluid having particles suspended therein and housed on a slidethat is positioned on the bed of the scanner 202. The scanner 200includes a modulation pattern 204 and a modulation pattern 206. Themodulation pattern 204 is moved relative to the particle 202 in a firstdirection 212 while the modulation pattern 206 is subsequently movedrelative to the particle 202 in a second direction 210. The first andsecond directions are, in one form, substantially perpendicular to oneanother. It should be appreciated that the bed (or a fixture) of thescanner 200 may also be moved to generate the relative motion betweenthe particle and the modulation pattern. It should be furtherappreciated that the modulation pattern 204 and 206 may be excitationpatterns to excite the particle, or optical elements provided tomodulate a constant light emission from the particle. The relativemovement contemplated will result in detection of light and acorresponding modulated signal that will allow for the determination ofthe location of the particle. In this regard, the location of theparticle may be determined at the position where both modulationpatterns yield a suitable signal.

Implementation of such a two dimensional analysis provides advantages.For example, this form of analysis results in a higher spatialresolution. In addition, an improved signal to noise ratio may beexperienced.

Further, the presently described embodiments have been describedprimarily in connection with optical methods of particlecharacterization, especially those involving visible light. However, itshould be appreciated that the presently described techniques andsystems may also be applied to other non-optical methods.

Referring to FIG. 15, an example of a non-optical method is one thatutilizes electrical properties (such as capacitance, conductance orimpedance) instead of light as a modulated output signal. In thisexample, a particle 400 may be moved down a channel 402 through anelectrode array 404, instead of an optical array. The electrode array404 is connected to a measurement device 406 and is set up to record thetime dependent signal (e.g. capacitance or current) influenced by themoving particle and operates to achieve the objectives of the presentlydescribed embodiments described herein.

The teachings of the presently described embodiments may be furtherextended. For example, in all cases where fluorescence intensity is weakand fluorescence particles are moving, this technique can be applied toincrease the signal-to-noise ratio. Examples:

Particle/Molecule counting, Cytometry: Counting fluorescent moleculesthat pass the modulated excitation region with high signal-to-noiseratio

Fluorescence spectroscopy: Measuring the fluorescence spectra ofparticles which pass through the modulated excitation region with highsignal-to-noise ratio by coupling the fluorescence light into aspectrometer. With reference to FIG. 14, a particle 600 travels down achannel 602. A shadow mask 604 creates a modulation on each individualpixel of the detector 608. Each pixel of detector 608 records the signalat a particular wavelength over a subrange Δ determined by the linearvariable filter 606. This system may be adjacent a particle detectorsystem 610 having a detector 612.

In accord with the presently described embodiments, relative motionbetween the particle and the spatially modulated excitation or emissionpattern is described. However, instead of moving the particle throughthe spatially modulated excitation pattern, the detection system canalso be scanned along a channel or carrier/chip. In the case of a chipthe particles of interest are fixed on the chip and, e.g., the absoluteposition of particles on the chip is determined.

The concept can, for example, also be applied to fluorescence read-outof a bio-chip.

Spatial modulations can be achieved in different manners. For example,geometry may provide a basis for spatial modulation. In this regard, aspatially modulated shadow mask, e.g. interdigitated finger-type mask, aspatially modulated phase mask, a micro-lens array or a micro-mirrorarray may be used.

Spatial modulation may also be achieved using electric or magneticfields. In this regard, emitted fluorescence intensity can be affectedby the modulated field. Also, the density of the fluorescence object maybe modulated by the field and the optical path can be affected by thefield.

Spatially modulated acoustic field (standing or moving acoustic waves,surface acoustic waves) may also be used. In this regard, emittedfluorescence intensity can be impacted by the modulated field. Thedensity of the fluorescence object may be modulated by the field. And,the optical path can be affected by the field.

Spatially modulated environment (e.g. stationary molecular coatings)within the moving path creating a spatially modulated fluorescencequenching may also be useful.

A spatially modulated micro-cavity which influences the emissionproperties of the moving object may likewise be applied to achieveobjectives of the presently described embodiments.

Advantages of the present invention are apparent. First, the location ofa particle can be determined with high resolution by analyzing the timedependence of a generated signal. This is enabled by a spatiallymodulated excitation pattern (e.g., interference pattern) in combinationwith a relative movement between a particle and excitation pattern.

Second, the lower bound of the spatial resolution is determined by thefeature size of an interference pattern which can be chosen much smallerthat 1 μm. Dependent upon the signal-to-noise ratio, the time coding ofthe signal, the relative speed of the particles and the pattern, thesampling rate of the detector, and the applied evaluation technique, itis feasible to achieve a spatial resolution better than the feature sizeof the interference pattern.

Third, the analyzed signal is modulated with periodic excitationvariation. Lock-in techniques or correlation techniques can be appliedto significantly enhance the signal-to-noise ratio.

Fourth, no critical optics are needed to focus the excitation light intoa very small volume or collect light out of a small volume.

Fifth, the techniques can be integrated into a lab-on-a-chip platformand can be easily extended to parallel multi-fluidic-channel analysis.

Sixth, several fluorescent particles which are within the interferencepattern can be detected simultaneously. The feature size of theinterference pattern determines the distance between two particles,which can be separated. Dependent upon the signal-to-noise ratio, thetime coding of the signal, the relative speed of the particles and thepattern, the sampling rate of the detector, and the applied evaluationtechnique, it is feasible to achieve a particle separation better thanthe feature size of the interference pattern.

Seventh, reduced intensity of the excitation light reduces damage, e.g.on living cells or bleaching of dyes.

It will be appreciated that various of the above-disclosed and otherfeatures and functions, or alternatives thereof, may be desirablycombined into many other different systems or applications. Also thatvarious presently unforeseen or unanticipated alternatives,modifications, variations or improvements therein may be subsequentlymade by those skilled in the art which are also intended to beencompassed by the following claims.

The invention claimed is:
 1. A method for characterizing particlescomprising; moving a particle within a channel; generating spatiallymodulated output light comprising moving the particle relative to ashadow mask disposed between the particle and a detector, the relativemotion causing light emission from the particle to be spatiallymodulated; recording a time modulated signal based on the spatiallymodulated output light, the time modulated signal including at leastthree transitions from an on state to an off state and includinginformation about a position of the particle within the channel; anddetecting and evaluating the time modulated signal.
 2. The method as setforth in claim 1 wherein the shadow mask is operative to modulate thesignal obtained from the particle as a function of a position of aparticle.
 3. The method as set forth in claim 1 further comprisingmoving the shadow mask.
 4. The method as set forth in claim 1 furthercomprising at least one of determining a location of the particle basedon the signal, counting particles based on the signal, and sortingparticles based on the signal.
 5. The method as set forth in claim 1wherein the detecting comprises detecting with a pixilated detector. 6.The method as set forth in claim 1 wherein the particle is a portion ofa DNA molecule or a molecule attached to the DNA molecule and the signalis used to determine DNA sequencing.
 7. The method as set forth in claim1 wherein the detecting comprises using a spectrometer to receive afluorescent spectrum of the particle.
 8. The method as set forth inclaim 1 wherein the shadow mask is operative to pattern the light basedon at least one of geometry, electric or magnetic field, fluorescencequenching, particle concentration, density, and acoustic standing wave.9. The method as set forth in claim 1, wherein-generating the spatiallymodulated output light comprises generating a spatially modulatedexcitation region that includes an excitation pattern having at leastthree transitions from an area of relatively higher excitation to anarea of relatively lower excitation.
 10. The method as set forth inclaim 9 wherein the generating of the excitation region comprisesgenerating a spatially modulated pattern based on at least one ofgeometry, fluorescence quenching, analyte concentration, and density.11. The method as set forth in claim 1 further comprising at least oneof determining a location of the particle based on the time modulatedsignal, counting particles based on the time modulated signal, andsorting particles based on the time modulated signal.
 12. The method asset forth in claim 1 wherein the detecting comprises detecting with apixilated detector.
 13. The method as set forth in claim 1 wherein theparticle is a portion of a DNA molecule or a molecule attached to theDNA molecule and the signal is used to determine DNA sequencing.
 14. Themethod as set forth in claim 1 wherein detecting comprises using aspectrometer to receive the fluorescent spectrum of the particle. 15.The method as set forth in claim 1 wherein detecting and evaluating thetime modulated signal comprises detecting the time modulated signal intwo-dimensions to locate the particle.
 16. A method for characterizingparticles comprising; moving a particle within a channel; generatingspatially modulated output light comprising moving the particle relativeto a shadow mask disposed between the particle and a detector, therelative motion causing light emission from the particle to be spatiallymodulated recording a time modulated signal based on the spatiallymodulated output light, the time modulated signal including at leastthree transitions from an on state to an off state and includinginformation about a position of the particle within the channel; anddetecting the time modulated signal.
 17. A system for characterizingparticles comprising; a channel through which a particle can move; ashadow mask configured to provide spatially modulated output light byspatially modulating light emission from the particle based on relativemotion between the particle and the optical element; and, a detectionsystem to record a time modulated signal based on the spatiallymodulated output light and evaluate the time modulated signal, the timemodulated signal including at least three transitions from an on stateto an off state and the time modulated signal including the at leastthree transitions including information about a position of the particlewithin the channel, wherein the shadow mask is disposed between theparticle and the detection system.
 18. The system as set forth in claim17 further comprising an anti-resonant waveguide operative to cause theparticle to emit light.
 19. The system as set forth in claim 17 whereinthe system is configured to determine a location of the particle basedon the signal, count particles based on the signal, and sort particlesbased on the signal.
 20. The system as set forth in claim 17 wherein thedetection system allows for a two-dimensional evaluation.
 21. The systemas set forth in claim 17 wherein: the shadow mask is operative to causethe particle to create time modulated signal comprises a spatiallymodulated excitation region that includes an excitation pattern havingat least three transitions from an area of relatively higher excitationto an area of relatively lower excitation; and the detection system isconfigured to record a time modulated signal generated by the particleas the particle moves through the excitation region and is excited uponexposure to the excitation region.
 22. The system as set forth in claim17 further comprising analyzer circuitry configured to perform at leastone of determining a location of the particle based on the timemodulated signal, counting particles based on the time modulated signal,and sorting particles based on the time modulated signal.